Antibacterial antibiotics AM31α, AM31β and AM31γ

ABSTRACT

This disclosure describes a novel class of antibiotics, three of which have been designed AM31α, AM31β, and AM31γ and are produced in a microbiological fermentation under controlled conditions using a new strain of Streptoverticillium netropsis.

CROSS REFERENCE TO RELATED APPLICATION

This application is a .Iadd.reissue application of our original patentNo. 3,987,029, dated Oct. 19, 1976, Ser. No. 559,998, filed Mar. 19,1975, which is a .Iaddend.continuation-in-part of our co-pendingapplication Ser. No. 436,008, filed Jan. 23, 1974, and now abandoned.

BRIEF SUMMARY OF THE INVENTION

This invention relates to new organic compounds and, more particularly,is concerned with novel antibiotics which may be represented by thefollowing structural formula: ##STR1## wherein R is hydrogen, formyl, oralkanoyl having up to 12 carbon atoms. The three antibiotics wherein Ris hydrogen, acetyl, or propionyl in the above formula have beendesignated AM31α, AM31γ, and AM31β, respectively, and are produced byfermentation. Those compounds wherein R is formyl or alkanoyl in theabove formula may be readily prepared by formylating or alkanoylatingantibiotic AM31α by standard methods well known in the art. The presentinvention includes within its scope the antibiotics in dilute form, ascrude concentrates, and in pure crystalline form. The structure of thesenew antibiotics as well as their spectral and chromatographic propertiesdifferentiate them from previously described antibacterial agents.

The novel antibiotics of the present invention form acid-addition saltswith a variety of pharmaceutically acceptable organic and inorganic saltforming reagents. Thus, acid-addition salts, formed by admixture of theantibiotic base with one, two, or three (when R is hydrogen) equivalentsiof an acid, suitably in a neutral solvent, are formed with such acidsas sulfuric, phosphoric, hydrochloric, hydrobromic, nitric, citric,lactic, tartaric, acetic, and related acids. For purposes of thisinvention, the antibiotic free bases are equivalent to their non-toxicacid-addition salts.

DETAILED DESCRIPTION OF THE INVENTION

The new antibiotics designated AM31α, AM31β, and AMB31γ are formedduring the cultivation under controlled conditions of a new strain ofStreptoverticillium netropsis. This new antibiotic producing strain wasisolated from a soil sample collected near Emma, Indiana. A viableculture of the new microorganism has been deposited with the CultureCollection Laboratory, Northern Utilization Research and DevelopmentDivision, United States Department of Agriculture, Peoria, Illinois, andhas been added to its permanent collection. It is freely available tothe public in this depository under its accession number NRRL 5774.

The description and identification of this new microorganism, maintainedin the culture collection of the Lederle Laboratories Division, AmericanCyanamid Company, Pearl River, New York as Culture No. Am31, wassupplied by Dr. H. D. Tresner of these laboratories. The following is ageneral description of the microorganism Streptoverticillium netropsis,based on diagnostic characteristics observed. Observations were made ofthe cultural, physiological and morphological features of the organismin accordance with the methods detailed by Shirling and Gottlieb (1).Descriptive details are recorded in Tables I-IV, and a generaldescription of the culture is given below. Underscored descriptivecolors were taken from Jacobson et al. (2).

Amount of Growth

Heavy on Bennett's, Kuster's Kuster'Hickey and Tresner's, Tomato PasteOatmeal and Pablum agars; good on Yeast Extract, Asparagine Dextrose,Benedict's and Rice agars; moderate on Potato Dextrose and light onCzapek's Solution agars.

Aerial Mycelium and Sporulation

Aerial mycelium pinkish-white, becoming Pearl Pink (3 ca) to Bisque (4ec) to Cork Tan (4 ie) in sporulating zones on most media.

Soluble Pigments

Yellowish to yellowish-brown or brownish on several media: none on YeastExtract, Inorganic Salts-Starch and Czapek's Solution Agars.

Reverse Color

Yellowish brown to brownish on most media.

Miscellaneous Physiological Reactions

Complete peptonization of purple milk; no curd formation: no nitratereduction in 14 days; gelatin completely liquefied in 7 days; only raceof melanoid pigments formed on petone-iron agar; NaCl tolerance in yeastExtract agar 7% but <10%. Carbon source utilization, according to thePridham and Gottlieb (3) method as follows: Good utilization ofi-inositol, glycerol, d-trehalose, and dextrose; poor to no utilizationof adonitol, l-arabinose, d-galactose, d-fructose, lactose, d-mannitol,d-meliboise, d-raffinose, l-rhamnose, salicin, sucrose, and d-xylose.

Micromorphology

Spore chains as regularly-spaced monoverticillate branches on aerialmycelium. Individual spore chains frequently terminating in hooks orcoils of one or two turns. Spores elongate, cylindrical. Most sporechains contain some phalangiform spores. Spores 0.5-0.6 μM × 1.0-1.2 μM.Spore surfaces smooth as determined by transmission electronmiscroscopy. Culture No. AM31 belongs to the genus Streptoverticilliumof the Actinomycetales. This genus is characterized by having its sporechains borne in whorls or verticils; there are about 40 membersrecognized. When compared with published descriptions (4) (5) as well aswith available reference specimens, Culture No. AM31 conforms mostclosely to the species concept for the taxon Streptoverticilliumnetropsis.

                                      TABLE I                                     __________________________________________________________________________    Cultural Characteristics of Streptoverticillium netropsis NRRL 5774           Incubation: 14 days                                                           Temperature 28° C                                                      Medium    Amount of Growth                                                                        Aerial Mycelium and/or Spores                                                                  Soluble Pigment                                                                        Reverse Color                                                                         Remarks                 __________________________________________________________________________    Yeast Extract                                                                           Good      Aerial mycelium pinkish-white                                                                  None     Lt. Amber                       Agar                becoming Bisque (4 ec) to (3 ie)                                              Cork Tan (4 ie) in sporulat-                                                  ing areas. Sporulation good.                              Asparagine Dex-                                                                         Good      Aerial mycelium pinkish-white                                                                  Yellowish-brown:                                                                       Cocoa Brown                     trose Agar          becoming Bisque (4 ec) to                                                                      light    (5 ni)                                              Cork Tan (4 ie) in sporula-                                                   ing areas. Sporulation good.                              Benedict's                                                                              Good      Aerial mycelium pinkish-white                                                                  Yellowish;                                                                             Lt. Amber                                                                             Abundant color-         Agar                becoming Bisque (4 ec) in                                                                      light    (3 ie)  less exudate on                             sporulating areas. Sporula-       colonies.                                   tion moderate.                                            Bennett's Heavy     Aerial mycelium pinkish-white                                                                  Yellowish-brown;                                                                       Cocoa Brown                                                                           Abundant yellow-        Agar                becoming Bisque (4 ec) to                                                                      light    (5 ni)  ish exudate on                              Cork Tan (4 ie) in sporulat-      colonies.                                   ing areas. Sporulation heavy.                             Inorganic Salts                                                                         Good      Aerial mycelium pinkish-white                                                                  None     Cork Tan                        Starch Agar         becoming Bisque (4 ec) to (4 ie)                                              Cork Tan (4 ie) in sporulat-                                                  ing areas. Sporulation good. -Kuster's                                                         Heavyake Aerial mycellium                                                              pinkish-white                                                                         Brownish; Lt.                                                                 Spice                   Agar                becoming Bisque (4 ec) to                                                                      moderate Brown                                               Cork Tan (4 ie) in sporulat-                                                                            (4 lg)                                              ing areas. Sporulation heavy.                             Czapek's Solution                                                                       Light     Aerial mycelium pinkish-white                                                                  None     Pearl Pink                      Agar                becoming Bisque (4 ec) in (3 ca)                                              sporulating areas. Sporula-                                                   tion light                                                Potato Dextrose                                                                         Moderate  Aerial mycelium pinkish-white                                                                  Yellowish-                                                                             Lt. Brown                                                                             Abundant color-         Agar                becoming Pearl Pink (3 ca) to                                                                  brown;   (4 ng)  less exudate on                             Bisque (4 ec) in sporulating                                                                   moderate         colonies                                    areas. Sporulation light                                  Hickey & Tresner's                                                                      Heavy     Aerial mycellium pinkish-white                                                                 Brown;   Dk. Wine                        Agar                becoming Bisque (4 cc) to                                                                      heavy    (7 pi)                                              Cork Tan (4 ie) in sporulat-                                                  ing areas. Sporulation heavy.                             Tomato Paste                                                                            Heavy     Aerial mycellium pinkish-white                                                                 Brown;   Cocoa Brown                                                                           Abundant yellow-        Oatmeal Agar        becoming Bisque (4 ec) to                                                                      heavy    (5 ni)  ish brownish-                               Cork Tan (4 ie) in sporulat-      trudate on                                  ing areas. Sporulation heavy.     colonies.               Pablum Agar                                                                             Heavy     Aerial mycelium pinkish-white                                                                  Brownish;                                                                              Lt. Spice                                           becoming bisque (4 ec) in                                                                      light    Brown                                               sporulating areas. Sporula-                                                                    light    (4 lg)                                              tion heavy.                                               Rice Agar Good      Aerial mycelium pinkish-white                                                                  Yellowish-                                                                             Lt. Brown                                           becoming Pearl Pink (3 ca) to                                                                  brown;   (4 ng)                                              Bisque (4 ec) in sporulating                                                                   light                                                        areas. Sporulation moderate.                              __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    Micromorphology of Streptoverticillium netropsis NRRL 6774                    Medium   Aerial Mycelium and/or Sporiferous Structures                                                        Spore Shape                                                                             Spore Size                                                                          Spore surface                 __________________________________________________________________________    Inorganic Salts-                                                                       Spore chains as regularly-spaced monoverti-                                                          Spores elongate,                                                                        0.5-0.6μM                                                                        Spore surfaces                Starch Agar                                                                            cillate branches on aerial mycellium.                                                                cylindrical. Most                                                                       ×                                                                             smooth as deter-                       Individual spore chains frequently termina-                                                          spore chains con-                                                                       1.0-1.2μM                                                                        mined by trans-                        ting in hooks or Coils of one or two turns.                                                          tain some phalangi-                                                                     mission electron                                                    form spores.    microscopy                    __________________________________________________________________________

                  TABLE III                                                       ______________________________________                                        Miscellaneous Physiological Reaction of Streptoverticillium                   netropsis NRRL 5774                                                           Temperature: 28° C                                                              Incubation                                                                               Amount of                                                                              Physiological                                    Medium   Period    Growth    Reaction                                         ______________________________________                                        Purple Milk                                                                            14 days   Good      Complete peptonization;                          Organic                      no curd remaining                                Nitrate Broth                                                                          7 days    Good      No nitrate reduction                             Organic                                                                       Nitrate Broth                                                                          14 days   Good      No nitrate reduction                             Gelatin  7 days    Good      Gelatin completely                               Peptone-iron                 liquified                                        Agar     48 hours  Good      Trace of melanoid                                Yeast Extract                pigments                                         Agar plus                                                                     (4, 7, 10 and                                                                 13% NaCl 10 days   Good      NaCl tolerance ≧                                                       7% but < 10%                                     ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Carbon Source Utilization Pattern of                                          Streptoverticillium netropsis NRRL 5774                                       Incubation 10 days                                                            Temperature: 28° C.                                                    Carbon Source         Utilization*                                            ______________________________________                                        Adonital              0                                                       l-Arabinose           0                                                       d-Galactose           0                                                       d-Fructose            0                                                       i-Inositol            3                                                       Lactose               0                                                       d-Mannitol            0                                                       Glycerol              3                                                       d-Melibiose           0                                                       d-Raffinose           0                                                       I-Rhamnose            0                                                       Salicin               1                                                       Sucrose               0                                                       d-Trehalose           3                                                       d-Xylose              0                                                       Dextrose              3                                                       Negative Control      0                                                       ______________________________________                                         *3 Good Utilization                                                           *2 Fair Utilization                                                           *1 Poor Utilization                                                           *0 No Utilization                                                        

It is to be understood that for the production of the new antibioticsAM31α, AM31β, and AM31γ, the present invention is not limited to thisparticular microorganism or to microorganisms fully answering thesegrowth and microscopic characteristics, which are given for illustrativepurposes only. In fact, it is desired and intended to include the use ofmutants produced from the described microorganism by various means suchas exposure to X-radiation, ultraviolet radiation, nitrogen mustard,actinophages, and the like.

Fermentation Process

Cultivation of the microorganism Streptoverticillium netropsis NRRL 5774may be carried out in a wide variety of liquid culture media. Mediawhich are useful for the production of these three antibiotics includean assimilable source of carbon such as starch, sugar, molasses,glycerol, etc., an assimilable source of nitrogen such as protein,protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.,and inorganic anions and cations, such as sodium, potassium, calcium,sulfate, phosphate, chloride, etc. Trace elements such as boron,polybdenum, copper, etc., are supplied as impurities of otherconstituents of the medium. Aeration in tanks and bottles is provided byforcing sterile air through or onto the surface of the fermentingmedium. Further agitation in tanks is provided by a mechanical impeller.An antifoaming agent, such as lard oil, may be added as needed.

Inoculum Preparation

Shaker flask inoculum of Streptoverticillium netropsis NRRL 5774 isprepared by inoculating 100 ml. of sterile liquid medium in 500 ml.flasks with scrapings or washings of spores from an agar slant of theculture. The following is an example of a suitable medium:

    ______________________________________                                        corn starch          24     gm.                                               bacto tryptone       5      gm.                                               Yeast extract        5      gm.                                               Beef extract         3      gm.                                               Glucose              1      gm.                                               Water to             1000   ml.                                               ______________________________________                                    

The pH is adjusted to 7.0 with NaOH.

The flasks are incubated at a temperature from 25°-29° C., preferably28° C., and agitated vigorously on a rotary shaker for 30 to 48 hours.These 100 ml. portions of inoculum are used to inoculate one and twelveliter batches of the same medium in 2 liter and 20 liter glassfermentors at 28° C. The inoculated mash is aerated with sterile airwhile growth is continued for 40 to 55 hours. These batches are used toinoculate tank fermentors.

Tank Fermentation

For the production of antibiotics AM31α, AM31β, and AM31γ in tankfermentors the following medium is preferably used:

    ______________________________________                                        Soy flour             40     gm.                                              Molasses              20     gm.                                              Glucose               10     gm.                                              Calcium carbonate     3      gm.                                              Water to              1000   ml.                                              ______________________________________                                    

Each tank is inoculated with 3 to 10% of inoculum made as describedabove. Aeration is supplied at the rate of 0.2 to 0.8 liter of sterileair per liter of broth per minute and the fermenting mixture is agitatedby an impeller driven at 200 to 400 rpm. The temperature is maintainedat 25°-29° C., usually at 28° C. The fermentaion is ordinarily continuedfor 80 to 100 hours, at which time the mash is harvested.

Isolation of AM31α, AM31β, and AM31γ as a Mixture

After the fermentation is completed, the harvested mash is filtered andthe filtrate, at pH 7.8, is passed through a 5 liter Amberlite IRC-50 (amethacrylic acid-divinyl benzene ion exchange resin) (NH₄ +) column at aflow rate of 250 ml./minute. After the column is washed with 25 litersof deionized water, the antibiotic activity is eluted with 30 liters of2N NH₄ OH and detected by the conventional disc agar diffusion assayagainst Klebsiella pneumoniae. The 30 liters of eluate at pH 11.7 isreduced to 2 liters and adjusted to pH 8.1 with 0.1N HCl. Theantibiotics in this concentrate are adsorbed onto an Amberlite CG-50 (amethacrylic acid-divinyl benzene ion exchange resin) (NH₄ +) column andeluted with 1.5N NH₄ OH. The column eluate is concentrated to 65 ml. ofan orange viscous syrup on a rotary evaporator. This concentrate ispassed through a Dowex 1-X2 (a trimethylbenzylammonium polystyrene crosslinked with 2% divinyl benzene) (OH⁻) (50-100 mesh) column and thecolumn is developed with water. The bioactive effluent is divided into 2major fractions based on visible color and bioactivity. One fraction (I).[.contans.]. .Iadd.contains .Iaddend.a mixture of AM31α, β, and.Iadd.γ.Iaddend. as a white powder that is essentially free of unwantedimpurities. The fraction, obtained in a liquid state because ofimpurities, may be further processed to yield more antibiotic mixture.This liquid fraction is passed through a Dowex 1-X2 (OH⁻) (200-400 mesh)column. The column is developed with water and 3 major fractions areobtained, two (II and IV) as a white powder and one (III) as a thickyellow syrup. This yellow syrup is further purified by adsorption on aDowex® 50X9 (a sulfonated polystyrene cross linked with 8% divinylbenzene) (H+) column. The column is rinsed with 250 ml. of water and theantibiotics are eluted with 500 ml. of 1.5N NH₄ OH. Two bioactivefractions are obtained in the 60-70 ml. and 290-700 ml. portions ofcolumn eluate. Each fraction is reduced to a small volume on a rotaryevaporator and then freeze-dried yielding a fine white powder (V) and awhite hygroscopic powder (VI). All of the fractions contain mixtures ofthe three components.

Separation of AM31α, AM31β, and AM31γ by Paper Chromatography

Components are differentiated from each other by paper chromatographyusing 1-butanol saturated with water to which 2% p-toluenesulfonic acidis added. The R_(f) values are: 0.61; 0.43; and 0.31, obtained byminhydrin.

In vitro Activity

The mixtue of antibiotics AM31α, β, and γ are active against a widevariety of gram positive and gram negative bacteria as determined by thestandard agar-well diffusion technique. The results of such a test onthe complex of the three components appear in Table V.

                  TABLE V                                                         ______________________________________                                                               Inhibition Zone                                          Name of Organism     (mm)*                                                  ______________________________________                                        Bacillas cereus (Waksman)                                                                            2.9                                                    Klebsiella pneumoniae  5.8                                                    (Friedlanders)                                                                Alcaligenes sp. ATCC 10153                                                                           4.1                                                    Bacillus subtilis                                                             (Stansly R-78)         5.8                                                    Bacillus subtilis (Resistant                                                                         1.0                                                    to Streptothricin)                                                            (Stansly R-76)                                                                Mycobacterium smegmatis                                                                              2.6                                                    (No. 607)                                                                     Staphylococcus aureus  2.5                                                    (resistant to tetracycline)                                                   Escherichia coli       4.0                                                    (Parke Davis)                                                                 Escherichia coli (resistant to                                                chloramphenicol)       7.3                                                    Staphylococcus aureus 209P                                                                           6.5                                                    (resistant to erythromycin)                                                   Corynebacterium serosis                                                                              8.5                                                    NRRL B-1397                                                                   Salmonella gallinarum No. 605                                                                        7.3                                                    Staphylococcus aureus (Smith)                                                                        5.3                                                    Klebsiella pneumoniae (AD)                                                                           9.2                                                    Pseudomonas aeruginosa 4.6                                                    ATCC 10145                                                                    Escherichia coli (Upjohn)                                                                            4.8                                                    Culture)                                                                      Aerobacter aerogenes   5.2                                                    Proteus mirabilis      1.6                                                    Salmonella iyphosa ATCC 6539                                                                         7.4                                                    Staphylococcus aureus  1.3                                                    ATCC 14154                                                                    Escherichia coli 311   5.0                                                    Pseudomonas aeruginosa PA7                                                                           3.6                                                    ______________________________________                                         *Zone value given as distance from edge of well to outer edge of              inhibition zone.                                                         

In vivo Results

the three antibacterial components AM31α, AM31β, and AM31γ are active invivo against a variety of organisms. These new antibacterials arethereby potentially useful as therapeutic agents in treating bacterialinfections in mammals. These new antibacterials can be expected to beusefully employed for treating or controlling bacterial infections byparenteral administration. The usefulness of these new antibacterialagents is demonstrated by their ability to control systemic lethalinfections in mice. A mixture of these three new antibiotics shows highin vivo antibacterial activity in mice against Escherichia coli,Salmonella typhosa and Klebsiella pneumoniae when administered by asingle subcutaneous dose to groups of Carworth Farms CF-1 mice, weighingabout 20 gm., infected intraperitoneally with 0.5 ml. of the indicatedbroth dilution of 5 hour cultures of the following organisms:Escherichia coli, 10⁻³ ; Salmonella typhosa undiluted; Klebsiellapneumoniae, 10⁻⁴. Table VI below, illustrates the in vivo antibacterialactivity of a mixture of AM31α, AM31β, and AM31γ against these threebacteria.

                  TABLE VI                                                        ______________________________________                                                         Alive/Total Mice Treated                                                      7 Days After Infection                                       ______________________________________                                        Single Subcutaneous Dose mg/kg.                                                                  Escherichia coli                                           512                2/2                                                        256                2/2                                                        128                2/2                                                         64                2/2                                                         32                0/2                                                        Infected non-treated controls                                                                     2/10                                                      Single Subcutaneous Dose Mg./kg.                                                                 Salmonella typhosa                                         512                2/2                                                        256                2/2                                                        128                2/2                                                         64                0/2                                                        Infected non-treated controls                                                                     0/10                                                      Single Subcutaneous Dose mg./kg.                                                                 Klebsiella pneumoniae                                      512                2/2                                                        256                2/2                                                        128                0/2                                                         64                0/2                                                        Infected non-treated controls                                                                     0/10                                                      ______________________________________                                    

This invention will be described in greater detail in conjunction withthe following specific examples.

EXAMPLE 1 Inoculum Preparation

A typical sterile medium used to grow the primary inoculum was preparedaccording to the following formula:

    ______________________________________                                        corn starch          24     gm.                                               bacto tryptone       5      gm.                                               Yeast extract        5      gm.                                               Beef extract         3      gm.                                               Glucose              1      gm.                                               Water to             1000   ml.                                               ______________________________________                                    

The pH was adjusted to 7.0 with NaOH

Washer of scraped spores from an agar slant of Streptoverticilliumnetropsis NRRL 5774 were used to inoculate two 500 ml. flasks containing100 ml. each of the above sterile medium. The flasks were placed on arotary shaker and agitated vigorously for 48 hours at 28° C. Theresulting flask inoculum was transferred to a 5 gallon glass fermentorcontaining 12 liters of the same sterile medium. The inoculcum mash wasaerated with sterile air while growth was carried out for 48 hours at28° C., after which the contents were used to seed a 300 liter tankfermentor.

EXAMPLE 2 Fermentation

A fermentation medium was prepared according to the following formula:

    ______________________________________                                        Soy flour             40     gm.                                              Molasses              20     gm.                                              Glucose               10     gm.                                              Calcium carbonate     3      gm.                                              Water to              1000   ml.                                              ______________________________________                                    

Twelve liters of inoculum, prepared as described in Example 1, were usedto inoculate 300 liters of the above sterilized fermentation medium. Thefermentation was carried out for 89 hours at 28° C. with an aerationrate of 0.5 liter of air/liter of mash/minute. The mash was agitated byan impeller driven at 300 rpm. The mash was harvested.

EXAMPLE 3 Isolation of a Mixture of AM31α, AM31β, and AM31γ

A 300 liter portion of whole harvest mash, prepared as described inExample 2, was filtered. The filtrate, having a pH of 7.8, was passedthrough a 5 liter Amberlite IRC-50 (NH₄ +) column, 4 inch × 60 inch, ata flow rate of 250 milliliters per minute. The column was washed with 25liters of deionized water. The antibiotic activity was eluted with 30liters of 2N NH₄ OH and detected by the disc agar diffusion assayagainst Klebsiella pneumoniae. The 30 liter eluate at pH 11.7 wasreduced to 2 liters and adjusted to pH 8.1 with 0.1N HCl. One liter ofthis concentrate was adsorbed on an Amberlite CG-50 (NH₄ ⁺) 3 × 52 cm.column and eluted with 500 ml. of 1.5N NH₄ OH. A duplicate run was madewith the remaining one liter concentrate and the elutes from the tworuns were combined and concentrated on a rotary evaporator to 65 ml. ofan orange viscous syrup. This 65 ml. concentrate was passed through aDowex 1-X2 (OH⁻) (50-100 mesh) 2 × 54 cm. column and the column wasdeveloped with water. The bioactive effluent was divided into twofractions based on visible color and bioactivity. One fraction wasrecovered from 735-1200 ml. of column effluent. It was freeze dried togive 132 mg. of a white powder (I). The other fraction was from 270-734ml. of column effluent. When this was freeze dried it remained in aliquid state, due to impurities. The liquid reaction was dissolved inwater and the solution was passed through a Dowex 1X2 (OH⁻) (200-400mesh) 1.5 × 21 cm. column. As the column was developed with water, 3major fractions were obtained with the indicated elution volumes:Fraction II, 55-114 ml.; Fraction IV, 36-775 ml.; and Fraction III,115-360 ml. of column effluent. Fractions II and IV were freeze-dried togive 4.76 gm. and 215 mg. of white powder, respectively. Fraction IIIwas concentrated to 75 ml. of yellow syrup which was adsorbed on a Dowex50-X8 (H⁺) 3 × 30 cm. column. This column was rinsed with 250 ml. ofwater and then eluted with 500 ml. of 1.5N NH₄ OH. Two bioactivefractions were obtained in the 60-70 ml. and 290-700 ml. portions of thecolumn effluent. Each fraction was reduced to a small volume on a rotaryevaporator, freeze-dried and the solids were recovered, yielding 685 mg.of fine white powder (Fraction V) and 6.78 gm. of white hygroscopicpowder (Fraction VI). Total yield, 12.572 gm.

Infrared and nuclear magnetic resonance spectra suggested that the abovefractions were all mixtures with approximately the same composition ofα, β and γ components.

Fraction II had [α]_(D) ²⁵° = + 85.0° ± 2.4° (C 0.41, H₂ O). Anal.Found: C, 42.03%; H, 8.08%; N, 10.47%. Nuclear magnetic resonance andinfrared spectra of a mixture of components are given in FIGS. 1 and 2,respectively, of the accompanying drawings.

EXAMPLE 4 Preparation of N-acetyl-O-trimethylsilyl Derivative ofAntibiotic AM31 Complex

The N-acetyl-O-trimethylsilyl derivative of the antibiotic AM31 complexwas prepared in the following way for mass spectrum characterization.Four milligrams of AM31 complex was mixed with 0.50 ml. of methaol and0.30 ml. of acetic anhydride. The solution was allowed to remainovernight at room temperature. The N-acetylated derivative wasprecipitated with 3-4 ml. of diethyl ether, washed several times withdiethyl ether and dried in a desiccator. The O-trimethylsilyl derivativewas then made by adding 0.5 ml. of TRI-SIL (a ready mix formulacontaining trimethylchlorosilane from Pierce Chemical Company, Rockford,Ill.). The silylation proceeded in a desiccator for 2 hours at roomtemperature after which the excess reagent was removed under vacuum. Theresidue was redistributed in benzene. The benzene-soluble material wasseparated from any residual solids and the solution was evaporated to aresidue in a stream of nitrogen. The data for the mass spectrum of theN-acetyl-O-trimethylsilyl derivative are given in Tables VII and IX. Themass spectrum of the degradation product of AM 31 complex,N-acetyl-O-trimethylsilyl derivative of the diaminodideoxyalditol isgiven in Table VIII.

                  TABLE VII                                                       ______________________________________                                        High Resolution Mass Spectral Data for N.Acetyl-O-                            Trmethylsilyl Derivative of AM31 Complex                                      Exact Mass                                                                    Observed     Calculated   Composition                                         ______________________________________                                        898.4324     898.4408     C.sub.36 H.sub.80 N.sub.3 O.sub.11 Si.sub.6         884.4238     884.4252     C.sub.35 H.sub.78 N.sub.3 O.sub.11 Si.sub.6         694.3380     694.3406     C.sub.28 H.sub.60 N.sub.3 O.sub.9 Si.sub.4          635.2995     635.3035     C.sub.26 H.sub.55 N.sub.2 O.sub.8 Si.sub.4          581.2909     581.2929     C.sub.23 H.sub.53 N.sub.2 O.sub.7 Si.sub.4          420.2056     420.2057     C.sub.17 H.sub.30 N.sub.1 O.sub.5 Si.sub.3          ______________________________________                                    

                  TABLE VIII                                                      ______________________________________                                        High Resolution Measurements of N-Acetyl-O-Trimethylsilyl                     Derivative of Diaminodideoxyalditol                                           (the degradation product of AM31 Complex)                                     Exact Mass                                                                    Observed     Calculated   Composition                                         ______________________________________                                        537.2668     537.2659     C.sub.21 H.sub.49 N.sub.2 O.sub.6 Si.sub.4          378.1952     378.1942     C.sub.15 H.sub.36 N.sub.1 O.sub.4 Si.sub.3          347.1822     347.1818     C.sub.14 H.sub.31 N.sub.2 O.sub.4 Si.sub.2          288.1450     288.1450     C.sub.12 H.sub.26 N.sub.1 O.sub.3 Si.sub.2          276.1421     276.1450     C.sub.11 H.sub.26 N.sub.1 O.sub.3 Si.sub.2          217.1081     217.1080     C.sub.9 H.sub.21 O.sub.2 Si.sub.2                   198.0949     198.0959     C.sub.9 H.sub.16 N.sub.1 O.sub.2 Si                 186.0934     186.0950     C.sub.8 H.sub.16 N.sub.1 O.sub.2 Si                 174.0929     174.0950     C.sub.7 H.sub.16 N.sub.1 O.sub.2 Si                 ______________________________________                                    

                  TABLE IX                                                        ______________________________________                                        Relative Abundance of Selected Ions Observed in Gas                           Chromatography/Mass Spectrum Analysis of                                      N-Acetyl-O-Trimethylsilyl Derivative of AM31 Complex                          α and γ Components                                                                  β Component                                                       Relative              Relative                                                Abundance             Abundance                                    Ion (m/e)  %          Ion (m/e)  %                                            ______________________________________                                        174        25         174        20                                           186        50         186        35                                           276        3.5        276        3.5                                          420        100        434        100                                          581        2.5        581        2.5                                          635        32.5       649        22.5                                         694        8.5        708        7.5                                          884        6.0        898        4.0                                          ______________________________________                                    

These spectra were obtained with a Varian CH7 Gas Chromatography/MassSpectrum with resolution. M/ΔM2000, ionizing voltage 70ev, and sourcetemperature 200° C. Gas chromatography conditions were as follows: Thecolumn for the gas chromatography was 6 feet long. The support was 0.7%OV-1 on glass Chrom Q (mesh size 100-200). The column temperature was230° C., injection port temperature 250° C., and detector temperature210° C. The carrier gas was nitrogen. The retention times of thecomponents were 24 minutes and 27.4 minutes.

EXAMPLE 5 Paper Chromatographic Separation of Antibiotic Components

The three components of the antibiotic mixture were differentiated using1-butanol saturated with water to which 2% p-toluenesulfonic acid wasadded. The R_(f) values for the components are 0.61, 0.43, and 0.31 asobtained by ninhydrin

EXAMPLE 6 Preparation and Identification of Diaminoalditol Fragment ofAntibiotic AM31 Complex

A 100 mg. sample of Antibiotic AM31 was heated in 5 ml. of 6N HCl for 16hours at 140° C. The resulting hydrolysate was filtered to removeconsiderable black precipitate, evaporated to a residue and dissolved inone ml. of water. This solution was poured onto a 1 × 2 cm. Dowex 1-X2(OH⁻) column (200-400 mesh), followed by 5 bed volumes of water. A darkbrown hygroscopic solid (33.9 mg.) was recovered on freeze-drying thecolumn effluent. A 10 mg. portion was used to prepare anN-acetyl-O-trimethylsilyl derivative for mass spectral studies asfollows: Ten mg. of the product was mixed with 1.25 ml. of methanol and0.75 ml. of acetic anhydride and allowed to remain overnight at roomtemperature. The N-acetylated compound was precipitated with 3-4 ml. ofdiethyl ether, washed several times with diethyl ether, dried in adesiccator and silylated with Tri-Sil (a ready mix formula of trimethylchlorosilane from Pierce Chemical Company, Rockford, Ill.). Thesilylation proceeded in a desiccator for 2 hours at room temperatureafter which the reagent was removed under vacuum and the residue wasredistributed in benzene. The benzene-soluble material was separatedfrom any residual solids and the solution was evaporated to a clearresin in a stream of nitrogen.

EXAMPLE 7 Preparation and Identification of Glucosamine Fragment ofAntibiotic AM31

A 5 mg. portion of antibiotic AM31 was dissolved in 3 ml. of 3N HCl andheated in a sealed vial at 100°-110° C. for 5 hours. The product wasevaporated to a residue which was redissolved in water and evaporated toremove HCl fumes. The residue was dissolved in 0.5 ml. of water andspotted onto sheets of Whatman No. 1 paper. The papergrams weredeveloped by the descending technique and the solvent allowed to dripoff the sheets. The AM31 hydrolysate had a component not differentiatedfrom glucosamine by mobility and color reactions in the followingsystems: 1-butanol:puridine:water (6:4:3), 11.0 cm. distance fromorigin; ethyl acetate:puridine:water (72:20:23), 1.5 cm. distance fromorigin. Zones were detached by ninhydrin and the Tollens reagent.

EXAMPLE 8 Preparation of Butyryl Derivative of AM31α

A solution of 10 g. of a mixture of AM31β and AM31γ, 11.3 g. ofdimedone, and 200 ml. of pyridine was refluxed for 7.5 hours, thenallowed to stand at room temperature overnight. The pyridine wasevaporated at reduced pressure and the residue was treated with 150 ml.of 1:1 methanol:water and 1.3 g. of dimedone and the resulting solutionwas refluxed for 6 hours. The solvent was removed completely at reducedpressure to yield a yellow gummy solid. Trituration with two 50 ml.portions of diethyl ether yielded, after air drying, 21.6 g. of yellowsolid.

The solid was dissolved in 250 ml. of 1:1 methanol:water and put on acolumn containing 350 ml. of Dowex 1-X2 (OH⁻) resin. The column waseluted with 1:1 methanol:water until no further product could be seen ona tlc plate (2.5 liters of eluent was used).

The eluate was put on a column containing 800 ml. of Dowex 50-X4 (H+)resin. The column was rinsed with 600 ml. of water, then eluted with 2%pyridine in water. The fraction of eluate between 2.5 and 5.5 literscontained the product. The solvent was removed at reduced pressure toyield a pale yellow gummy froth. This is the bis-dimedone derivative(II). ##STR2##

The froth was dissolved in 200 ml. of water, treated with 25 g. ofbarium hydroxide hydrate and the mixture was refluxed 1.25 hours. Themixture was filtered and the filtrate was treated with carbon dioxide topH 6.0. The mixture was filtered, the filter cake was washed with about100 ml. of water and the filtrate was put on a column of 700 ml. ofDowex 50-X4 (H⁺). The column was washed with one liter of water, 2liters of 2% pyridine in water, and then eluted with 3 liters of 2Nammonium hydroxide. The eluate was evaporated at reduced pressure toyield a yellow froth (9.8g.). The froth was lyophilized to yield 8.9 g.of very low density cream colored solid, the protected intermediate(III).

A solution of 11.8 g. of the protected intermediate (III) in 400 ml. ofmethanol was cooled to 5° C. and treated with 20 ml. of butyricanhydride. The mixture was stirred at 0° C. for one hour, then at roomtemperature for 16 hours. The solution was concentrated at reducedpressure, the residue was dissolved in 300 ml. of 1:1 methanol:water andput on a 700 ml. column of Dowex 50-X4 (H.sup.). The column was washedwith 2 liters of 1:1 methanol:water, then eluted with 2% pyridine inwater. The product (IV) is eluted in the fraction between 2.5 and 4.5liters of eluate. The eluate is evaporated at reduced pressure and theresidual gum is hydrolyzed without further purification. ##STR3## Theprotected antibiotic (IV) (about 12 g.) was dissolved in 400 ml. ofwater, cooled to 0°-5° C., and treated with a prechilled (to 5° C.)solution of 3% chlorine in carbon tetrachloride. The mixture was treatedwith 50 ml. of methanol and stirred at 0°-5° C. for 0.5 hour. The layerswere separated and the aqueous portion was washed with carbontetrachloride (100 ml.), then with chloroform (2×100 ml.), and treatedwith concentrated ammonium hydroxide to pH 5.0 (required about 5 ml.).This solution was treated with a solution of sodium bisulfite in waterto remove traces of chlorine (until no starch iodide test was obtained).Then retreated with ammonium hydroxide to pH 5.0 (required 4 ml. more).The solution of the crude antibiotic (V) was evaporated at reducedpressure to 25-50 ml. volume and submitted for chromatography.

EXAMPLE 9 Purification of Butyryl Derivative (V) of AM31

The solution (10 ml.) of the crude antibiotic (V) was placed onto acolumn (.15×22 cm.) of Amberlite XAD-2. The column was then eluted with250 ml. of each of the following solvents: methanol, 50% aqueousmethanol, and water. The antiobiotic eluted from the column in 12-144ml. of effluent and was detected by a paper disc agar:diffusion assaywith Kiebsiella pneumonia as a test organism. This fraction wasevaporated in vacuo to a residue which was dissolved in water and freezedried to obtain 8.6 g. of white powder. Seven grams of this material wasdissolved in a small volume of water and adsorbed onto a column (2×32cm.) of Dowex 50-X8 (NH4+) 50-100 mesh. The column was rinsed with 250ml. of water and then eluted with 250 ml. of 1.5N NH₄ OH as 7 ml.fractions were collected. The antibiotic, detected in reactions 32-67 bythe agar disc assay, was recovered by evaporaing the pooled fractions invacuo to a residue which was dissolved in water and freeze-dried toobtain 475 mg. of white powder. The antibiotic was identified by themass spectrum of an N-acetyl-O-trimethylsilyl derivative which hadcharacteristic ions at m/e 912 (M⁺ -15), 722, 663, and 448. The nmrspectrum had δ 1.01 (CH₃, t), 1.78 (CH₂, m), and 2.44 (CH₂, t) for thebutanoyl group along with signals expected for the other parts of themolecule.

The in vitro activity of this butyryl derivative was similar to that ofAM31 β and it protected mice against lethal infections of Klebsiellapneumonia at 256 mg./kg. of body weight.

EXAMPLE 10 Preparation of the Octanoyl and Dodecanoyl Derivatives ofAM31α

The octanoyl and dodecanoyl derivatives of AM31α were prepared by theprocedure of Example 8 except that caprylic anhydride and lauricanhydride, respectively, were employed in place of the butyric anhydrideof that examle. These derivatives possessed in vitro activity butsignificantly less (about 1/50) than that of AM31β .

.[.EXAMPLE 11.]. .[.Preparation of AM31β from the Daiminoalditol.].

.[.The N,N'-diethoxycarbonyldiaminoalditol (VI) was prepared from thediaminoalditol (obtained from the antibiotic hydrolyzate) by a procedureessentially the same as that employed by Nishimura et al (1) to prepareN,N'-diethoxycarbonyl-2-deoxystreptamne. To a solution of 5.0 g. of (VI)in dry dimethylformamide was added 500 mg. of anhydrousp-toluenesulfonic.]. ##STR4## .[.acid and 10 ml. of dry1,1-dimethoxycyclohexane. The resulting solution was stirred at 50° C.in vacuo (33 Torr.) for two hours and then evaporated to a residue whichwas mixed vigorously with ethyl acetate and a saturated aqueous solutionof barium hydroxide. The organic layer was separated, washed with waterand evaporated to the crudemono-O-cyclohexylidene-N,N-diethoxycarbonyldiaminoalditol (VII), weight5.2 g. A solution of (VII) (1.0 g.) in 20 ml. of dry benzenedioxane(2:1) was mixed with 2.0 g. of Drierite, 2.0 g. of dried mercuriccyanide, and 2.0 g. of3,4,6-tri-O-acetyl-N-p-methoxybenzylidene-α-D-glucosaminyl bromide (2)and the mixture was stirred at room temperature for 5 hours. TheDrierite was removed by filtration and the filtrate was evaporated to anoil which was dissolved in chloroform, washed with aqueous NaHCO₃ andwith water, and then evaporated to a viscous oil consisting of thedesired condensation product (VII), other isomers, and impurities

.[.The viscous oil containing (VIII) was dissolved in 5 ml. of methanoland 2.5 ml. of 50% acetic acid was added. This solution was heated on asteam bath for one hour and evaporated to an oil which was dissolved in100 ml. of methanol. This solution was cooled in an ice bath, 30 ml. ofpropionic anhydride was added, and the reaction mixture then allowed tostand at room temperature overnight. The reaction mixture was thenevaporated to a syrup which was then stirred overnight at roomtemperature with 100 ml. of saturated aqueous barium hydroxide in asealed flask. The excess barium hydroxide was removed by precipitationwith CO₂. The filtrate contained the antibiotic AM31β which could bepurified by ion exchange chromatography on Amberlite IRC-50 (NH₄ ⁺) orDowex 50-X8(NH₄ ⁺) as previously described for the naturalantibiotic..].

We claim: .[.1. A compound selected from the group consisting of thoseof the formula: ##STR6## wherein R is selected from the group consistingof hydrogen, formyl, and alkanoyl having up to 12 carbon atoms; and thepharmaceutically acceptable acid-addition salts thereof..]. .[.2. Acompound according to claim 1 wherein R is hydrogen..]. .[.3. A compoundaccording to claim 1 wherein R is formyl..]. .[.4. A compound accordingto claim 1 wherein R is acetyl..]. .[.5. A compound according to claim 1wherein R is propionyl..]. .[.6. A compound according to claim 1 whereinR is butyryl..]. .[.7. A compound according to claim 1 wherein R isoctanoyl..]. .[.8. A compound acording to claim 1 wherein R isdodecanoyl..]. .[.9. The antibiotic free base according to claim 2..]..[.10. The antibiotic free base according to claim 3..]. .[.11. Theantibiotic free base according to claim 4..]. .[.12. The antibiotic freebase according to claim 5..]. .[.13. The antibiotic free base accordingto claim 6..]. .[.14. The antibiotic free base according to claim 7..]..[.15. The antibiotic free base according to claim 8..]. .Iadd.
 16. Acompound selected from the group consisting of the antibiotic free baseof the formula: ##STR7## and the pharmaceutically acceptableacid-addition salts thereof. .Iaddend..Iadd.
 17. A compound selectedfrom the group consisting of the antibiotic free base of the formula:##STR8## and the pharmaceutically acceptable acid-addition saltsthereof. .Iaddend.